The present invention relates to novel molecules, such as proteins, polypeptides and nucleotides, involved in the transduction of signals in the hedgehog-patched (HH-PTC) pathway which takes place during the development of the cells of a human body. The invention also relates to certain advantageous uses of the molecules according to the invention in therapy and diagnosis.
In the study of the development of cells, fruit flies have extensively been used as a model, as they are less complex than mammalian cells.
Pattern formation takes place through a series of logical steps, reiterated many times during the development of an organism. Viewed from a broader evolutionary perspective, across species, the same soil of reiterative pattern formations are seen. The central dogma of pattern formation has been described (Lawrence and Struhl, 1996). Three interlocking and overlapping steps are defined. Firstly, positional information in the form of morphogen gradients allocate cells into non-overlapping sets, each set founding a compartment. Secondly, each of these compartments acquire a genetic address, as a result of the function of active xe2x80x9cselectorxe2x80x9d genes, that specify cell fate within a compartment and also instruct cells and their descendents how to communicate with cells in neighboring compartments. The third step involves interactions between cells in adjacent compartments, initiating new morphogen gradients, which directly organize the pattern.
Taking these steps in greater detail, one finds the first step in patterning to be the definition of sets of cells in each primordium. Cells are allocated according to their positions with respect to both dorsoventral and anterior/posterior axes by morphogen gradients. Allocation of cells in the dorsoventral axis constitutes the germ layers, such as mesoderm or neurectoderm.
In segmentation, the second step (the specification of cell fate in each compartment) is carried out by the gene engrailed and elements of the bithorax complex. Engrailed defines anterior and posterior compartments both in segmentation and in limb specification.
The third step in pattern formation, secretion of morphogens, functions to differentiate patterns within compartments (and thereby establish segment polarity). Initially, all cells within a compartment are equipotent, but they become diversified to form pattern. Pattern formation depends on gradients of morphogens, gradients initiated along compartment boundaries. Such gradients are established by a short-range signal induced in all the cells of the compartment in which the above mentioned selector gene engrailed is active. For segment polarity, this signal is Hedgehog. In the adjacent compartment the selector gene is inactive, ensuring that the cells are sensitive to the signal. The Hedgehog signal range is probably only a few rows of cells wide; responding cells become a linear source of a long-range morphogen, that diffuses outward in all directions. There are three known Hedgehogs, Sonic (SHH), Indian (IHH) and Desert (DHH). The proteins they encode can substitute for each other, but in wildtype animals, their distinct distributions result in unique activities. SHH controls the polarity of limb growth, directs the development of neurons in the ventral neural tube and patterns somites. IHH controls endochondral bone development and DHH is necessary for spermiogenesis. Vertebrate hedgehog genes are expressed in many other tissues, including the peripheral nervous system, brain, lung, liver, kidney, tooth primordia, genitalia and hindgut and foregut endoderm.
Thus, segment polarity genes have been identified in flies as mutations, which change the pattern of structures of the body segments. Mutations in these genes cause animals to develop the changed patterns on the surfaces of body segments, the changes affecting the pattern along the head to tail axis. For example, mutations in the gene patched cause each body segment to develop without the normal structures in the center of each segment. Instead there is a mirror or image of the pattern normally found in the anterior segment. Thus, cells in the center of the segment make the wrong structures, and point them in the wrong direction with reference to the over all head-to-tail polarity of the animal.
About sixteen genes in the class are known. The encoded proteins include kinases, transcription factors, a cell junction protein, two secreted proteins called wingless (WG) and the above mentioned Hedgehog (HH), a single transmembrane protein called patched (PTC) and some novel proteins not related to any known protein. All of these proteins are beleived to work together in signaling pathways that inform cells about their neighbors in order to set cell fates and polarities.
PTC has been proposed as a receptor for HH protein based on genetic experiments in flies. A model for the relationship is that PTC acts through a largely unknown pathway to inactivate both its own transcription and the transcription of the wingless segment polarity gene. This model proposes that HH protein, secreted from adjacent cells, binds to the PTC receptor, inactivates it and thereby prevents PTC from turning off its own transcription or that of wingless. A number of experiments have shown coordinate events between PTC and HH.
WO 96/11260 discloses the isolation of patched genes and the use of the PTC protein to identify ligands, other than the established ligand Hedgehog, that bind thereto. However, even though it is briefly suggested that drugs may be identified which can prevent the transduction of signals by the PTC protein, there are no teachings as regards how such signals are transduced.
In order to elucidate how the Hedgehog elecits signal transduction, a large complex containing the kinesin-related protein costa 12 has been proposed (Robbins et al; Cell: Jul. 25, 1997, 90(2), p. 225-34). Said complex includes the products of at least three genes: fused (a protein-serine/threonine kinase), cubitus interruptus (a transcription factor) and costal2 (a kinesin-like protein). It is concluded that in Drosophila, the complex may facilitate signaling from HH by governing access of the cubitus interruptus protein to the nucleus.
Therond et al have also studied signaling from Hedgehog in Drosophlila (Proc. Natl.Acad. Sci. USA, Apr. 30, 1996, 93(9), p. 4224-8). The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. It is proposed that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. As a conclusion, this reference suggests, that a reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which the hedgehogs acts.
The gene encoding a suppressor of the mutant phenotype of the above discussed fused gene, denoted suppressor of fused (SUFU), has been studied in Drosophila and mice. It has been shown by mRNA levels analysed by in situ hybridisation that during mouse embryogenesis, the mouse suppressor of fixed (MSUFU) is expressed. This occurs in a specific pattern on top of a wide spread basal level including several tissues; at specific sites during limb development, in developing lung, genital area, skeletal development in somites and ribs, etc. The expression pattern is similar to the mouse patched gene (MPTC) and is compatible with a role for MSUFU in the HH-MPTC, such as SHH-PTC, signaling pathway. Thus, even though the gene suppressor of fused (SUFU) has been identified in Drosophila and studied in mice, it has not been isolated from human beings before. The prior art teachings about the interactions between the hedgehog ligand and the patched receptor in mammals is not at all sufficient for practical applications in regard of human beings. Firstly, there is a need of a better understanding of how the signals are actually elicited and transduced in said pathway. Secondly, the human homologues of the genes implicated in this pathway must also be isolated, and the cDNA thereof identified, in order to enable recombinant reproduction thereof. However, due to the substantial genetic differences between fruit flies and human beings and the complex nature of the human genome, the isolation of the human homologues to genes identified in the fruit flie is in no way a straightforward task.
The present invention fulfill the above defined need by providing novel human homologues to molecules associated with the Drosophila suppressor fused (SUFU) gene and implicated in the transduction of the signals elicited by the interaction between the patched receptor (PTC, sometines denoted the NBCCS gene) and any one of the hedgehog ligands, e.g. the sonic hedgehog (SHH). The molecules according to the invention are nucleic acids as well as polypeptides and proteins encoded thereof, which are useful within several fields, including the study of different conditions, such as cancer and development of cancer therapies, hereditary malformation syndromes the regulation of gene transcription, studies of embryonic development, tissue repair etc. These applications will be discussed in more detail below.
The proposed research will give important basic understanding of a singaling pathway that is central to normal development and often disrupten in disease. The new knowledge will also be of great value when considering new therapeutic strategies involving modification of SHH-PTCH singaling. Potential areas include tissue repair/wound healing (brain, bone, cartilage, skin), neurodegenerative disease, testicular function and cancer. We also foresee a use of the SU(FU)H gene/protein as a diagnostic tool.
Recently, the present inventors have shown that SU(FU) co-localizes with alpha-tubulin in human cells, whereby it is associated with microtubuli similar to FU and cos2 in Drosophila.
The novel SUFU gene according to the present invention encodes a protein which is expressed in all adult tissues tested by the inventors. However, during embryonic development, it has a very restricted expression pattern, which partially overlaps with the expression of other members of the hedgehog signalling pathway and tendentially congrues with the expression of the Ci-homolog Gli-3 rather than Gli-1. In reporter gene assays, SUFU according to the invention was shown to downregulate Gli-1 induced expression.